Previous work has determined that contraction of venous smooth muscle is at least in part regulated by phosphorylation of the 20000 dalton myosin light chain (LC20) and bears some mechanistic similarities to other smooth muscle types. The long range objective of this proposal is to determine if the mechanism of phosphorylation can fully explain the regulation of venous smooth muscle contraction by various vasoactive stimuli. Isolated segments of canine jugular and femoral or human saphenous veins incubated in tissue baths will be assayed during contraction for rates of crossbridge cycling. They will also be quick frozen and analyzed for LC20 phosphorylation on a 2-dimensional isolectric focusing - SDS electrophoretic gel system. Some experiments will use tissues which are chemically skinned to allow direct control of ions in the intracellular space. Specific questions to be asked include: 1) Is phosphorylation uniquely associated with elevated levels of crossbridge cycling or can these parameters be uncoupled? 2) To what extent does the putative transient in myoplasmic calcium influence the course of LC20 phosphorylation during contraction and what is its intracellular mechanism? 3) Is LC20 phosphorylation alone capable of contracting venous smooth muscle? 4) Even if phosphorylation itself induces contraction, can calcium ion produce additional tension development or maintenance by acting at a second site on the myofilament? If so, what is its relationship to LC20 phosphorylation? This work will have a bearing on theories which ascribe a role for venous contractility in the formation of autocoid-induced lesions which are potential sites for thrombus formation. Studies in this area have used canine jugular and femoral veins as a model. The use of human saphenous veins which are employed extensively in bypass surgery will increase understanding of venous contractile regulation in man.